1. Field of the Invention
This invention relates to methods for detecting tumor-derived or tumor-associated mammalian ribonucleic acid (RNA) in bodily fluids such as blood plasma and serum obtained from an animal, most preferably a human. Specifically, the invention is directed towards methods for detecting RNA in bodily fluids from a human bearing a premalignant lesion or a malignancy, ranging in severity from localized neoplasia to metastatic disease. The methods of the invention are particularly drawn to detecting RNA encoding all or a portion of particular genes associated with neoplastic growth, development, or pathogenesis. In particular, these methods are drawn to genes associated with tumor growth factors such as tyrosine kinase mediated growth factors (for example, epidermal growth factor, EGF) and their receptors (for example, epidermal growth factor receptor (EGFr), and her-2/neu), as well as oncogenes such as c-myc oncogene. The methods of the invention are further particularly drawn to detecting RNA derived or associated with tumor-associated ribonucleoprotein, such as but not limited to heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) and associated ribonucleoprotein. In view of the essential role of RNA in expressing genes and producing proteins encoded thereby, detection and monitoring of RNA provides a convenient and reliable method for assessing and monitoring gene expression associated with neoplastic disease, thereby enabling the detection, diagnosis, monitoring, evaluation, and prognosticating of cancer and premalignancy.
2. Background of the Related Art
The pathogenesis and regulation of cancer is dependent upon gene expression, comprising production and translation of RNA to produce proteins involved with a variety of cellular processes, such as cell proliferation, regulation, and death. Furthermore, some gene expression, resulting in the existence of RNA and the proteins translated therefrom in cells and tissues, although not necessarily involved in neoplastic pathogenesis or regulation, may comprise a phenotype of recognizable characteristics for particular neoplasms, for example, either by being expressed at elevated levels or by being inappropriately expressed in said cells or tissues.
Tyrosine kinase-mediated growth factors and their receptors such as epidermal growth factor (EGF), epidermal growth factor receptor (EGFr), and her-2/neu, play important roles in the growth of many epithelial cancers and their response to insult or injury. Oncogenes such as c-myc play important roles in the pathogenesis of many cancers. Other proteins, such as hnRNP A2/B1 and associated ribonucleoproteins including hnRNP A2 (heterogeneous nuclear ribonucleoprotein A2) and hnRNP B1 (heterogeneous nuclear ribonucleoprotein B1) are overexpressed early in the development of some cancers. Detection of RNA encoding EGF, EGFr her-2/neu, c-myc or hnRNP A2/B1 provides a method for detecting and monitoring a wide spectrum of cancers and premalignancies, and can have prognostic significance. Tyrosine kinase-mediated growth factors and their receptors further provide potential targets for cancer therapies such as monoclonal antibody-based therapies (for example, herceptin for her-2/neu and CA-225 for EGFr), small molecule therapies and tyrosine kinase inhibitors, as well as vaccine therapies. Detection of EGF, EGFr and her-2/neu RNA can thus provide methods for selecting and monitoring patients for such therapies.
RNA associated with cancer and premalignant or neoplastic states, such as RNA encoding EGF, EGFr her-2/neu, c-myc or hnRNP A2/B1 are referred to herein as tumor-derived or tumor-associated RNA. Co-owned and co-pending U.S. patent application Ser. No. 09/155,152, incorporated by reference herein in its entirety, provides methods by which mammalian tumor-associated or tumor-derived RNA in bodily fluids such as plasma and serum can be detected and utilized for detecting, monitoring, or evaluating cancer or premalignant conditions. U.S. patent application Ser. No. 09/155,152, incorporated by reference herein in its entirety, further taught that tumor-associated or tumor-derived RNAs include erb-B-1 mRNA (also known as epidermal growth factor receptor mRNA), her-2/neu MRNA (also known as erb-B-2 mRNA), c-myc mRNA, and hnRNP A2/B1 associated RNA were advantageously detected in bodily fluids such as blood plasma or serum.
RNA encoding EGF, EGFr, her-2/neu, c-myc, and hnRNP A2/B1 being recognized as tumor-associated RNAs, there is a newly-appreciated need in the art to identify premalignant or malignant states characterized by said RNA in animals including humans by detecting said RNA in bodily fluids such as blood plasma or serum.
The present invention provides methods for detecting EGF RNA, EGFr RNA, her-2/neu RNA, and hnRNP A2/B1 RNA, or any combination thereof, in bodily fluids, preferably in blood and most preferably in blood plasma and serum, and in other bodily fluids including but not limited to urine, effusions, ascites, saliva, cerebrospinal fluid, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, sputum and bronchial secretions, and breast fluid and associated lavages and washings. The inventive methods comprise detecting extracellular mammalian tumor-associated or tumor-derived RNA such as EGF RNA, EGFr RNA, her-2/neu RNA c-myc RNA or hnRNP A2/B1 RNA, or any combination thereof, in said bodily fluids.
In preferred embodiments, the methods of the invention comprise the step of amplifying and detecting extracellular EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, and/or hnRNP A2/B1 RNA or any combination thereof from bodily fluids of an animal, most preferably a human.
In particularly preferred embodiments, the present invention provides methods for detecting EGF RNA, or EGFr RNA, or her-2/neu RNA, or c-myc RNA, or hnRNP A2/B1 RNA, or any combination thereof in blood or a blood fraction, including plasma and serum, and other bodily fluids. In these embodiments, the method comprises the steps of extracting mammalian RNA from blood, plasma, serum, or other bodily fluid, wherein a fraction of the extracted RNA comprises extracellular EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, or hnRNP A2/B1 RNA; or any combination thereof; in vitro amplifying RNA or cDNA corresponding thereto encoding EGF, EGFr, her-2/neu, c-myc, or hnRNP A2/B1 or any combination thereof; and detecting the amplified products produced from said mRNA or cDNA.
In a first aspect of this embodiment, the present invention provides methods for detecting EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, hnRNP A2/B1 or any combination thereof in blood or blood fractions, including plasma and serum, in an animal, most preferably a human. Said methods advantageously permit detection, diagnosis, monitoring, treatment, or evaluation of proliferative disorders, particularly stages of neoplastic disease, including premalignancy, early cancer, non-invasive cancer, carcinoma in-situ, invasive cancer, metastatic cancer and advanced cancer, as well as benign neoplasms. In this aspect, the method comprises the steps of extracting mammalian RNA from blood or blood plasma or serum, in vitro amplifying qualitatively or quantitatively a fraction of the extracted RNA or the corresponding cDNA wherein said fraction comprises EGF-, EGFr-, her-2/neu-, c-myc-, or hnRNP A1/A2-encoding RNA or combination thereof, and detecting the amplified products of said RNA or cDNA.
The invention in a second aspect provides methods for detecting EGF-, EGFr-, her-2/neu, -c-myc-, or hnRNP A2/B1-encoding RNA or any combination thereof in any bodily fluid. Preferably, said bodily fluid is whole blood, blood plasma, serum, urine, effusions, ascitic fluid, amniotic fluid, saliva, cerebrospinal fluid, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions including sputum, secretions or washings from the breast, and other associated tissue washings from an animal, most preferably a human. In this aspect, the method comprises the steps of extracting mammalian RNA from the bodily fluid; in vitro amplifying in a qualitative or quantitative fashion a fraction of the extracted RNA, wherein said fraction comprises extracellular EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, hnRNP A2/B1 RNA or any combination thereof, or more preferably cDNA corresponding thereto, and detecting the amplified product of said RNA or cDNA.
In these embodiments, the inventive methods are particularly advantageous for detecting, diagnosing, monitoring, treating, or evaluating proliferative disorders in an animal, most preferably a human, said proliferative disorders particularly including stages of neoplastic disease, including premalignancy, early cancer, non-invasive cancer, carcinoma-in-situ, invasive cancer, metastatic cancer and advanced cancer as well as benign neoplasm.
Thus, in another aspect the invention provides methods for evaluating an animal, most preferably a human, for premalignant or malignant states, disorders, or conditions. The inventive methods comprise detecting extracellular mammalian tumor-associated or tumor-derived RNA including EGF RNA, EGFr RNA, her-2/neu RNA c-myc RNA and hnRNP A2/B1 RNA or any combination thereof in bodily fluids, preferably blood and most preferably blood plasma and serum as well as in other bodily fluids, preferably urine, effusions, ascites, amniotic fluid, saliva, cerebrospinal fluid, cervical, vaginal, and endometrial secretions, gastrointestinal secretions, bronchial secretions, breast fluid, and associated tissue washings and lavages.
The methods of the invention are also useful for identifying EGF-, EGFr-, her-2/neu-, c-myc-, or hnRNP A2/B1-expressing cells or tissue in an animal, most preferably a human. In these embodiments, detection of an in vitro amplified product of EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, or hnRNP A2/B1 RNA or cDNA corresponding thereto using the methods of the invention indicates the existence of EGF, EGFr, her-2/neu, c-myc, or hnRNP A2/B1-expressing cells or tissue in a human.
The invention further provides diagnostic kits for detecting EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, hnRNPA2/B1 RNA or any combination thereof in bodily fluid, preferably blood plasma or serum, wherein the kit comprises oligonucleotide primers, probes, or both primers and probes for amplifying and detecting said EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, hnRNPA2/B1 RNA or any combination thereof or cDNA derived therefrom. In advantageous embodiments, the kit may further comprise instructions and reagents for performing methods for extracting RNA from the bodily fluid, reverse-transcribing said RNA into cDNA or reagents for performing in vitro amplification.
In preferred embodiments of the inventive methods, EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, hnRNP A2/B1 RNA or any combination thereof is extracted from whole blood, blood plasma or serum, or other bodily fluids using any effective extraction method including but not limited to gelatin extraction methods; silica, glass bead, or diatom extraction methods; guanidinium thiocyanate acid-phenol based extraction methods; guanidinium thiocyanate acid based extraction methods; methods using centrifugation through cesium chloride or similar gradients; phenol-chloroform based extraction methods; or other commercially available RNA extraction methods. In this aspect of the invention, RNA is extracted from plasma, serum, or other bodily fluid. In other aspects of the invention, extraction may alternatively be performed using probes that specifically hybridize to a particular RNA.
In preferred embodiments of the inventive methods, EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, hnRNP A2/B1 RNA or any combination thereof, or more preferably cDNA derived therefrom is amplified using an in vitro amplification method such as reverse transcriptase polymerase chain reaction (RT-PCR); ligase chain reaction; DNA signal amplification; amplifiable RNA reporters; Q-beta replication; transcription-based amplification; isothermal nucleic acid sequence based amplification; self-sustained sequence replication assays; boomerang DNA amplification; strand displacement activation; cycling probe technology; or any combination or variation thereof.
In preferred embodiments of the inventive methods, amplification products of EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, hnRNP A2/B1 RNA or any combination thereof, or more preferably cDNA produced therefrom, are detected using a detection method such as gel electrophoresis; capillary electrophoresis; conventional enzyme-linked immunosorbent assay (ELISA) or modifications thereof, such as amplification using biotinylated or otherwise modified primers; nucleic acid hybridization using specific labeled probes, such as fluorescent-, radioisotope-, or chromogenically-labeled probes; Southern blot analysis; Northern blot analysis; electrochemiluminescence; laser-induced fluorescence; reverse dot blot detection; and high-performance liquid chromatography.
In particularly preferred embodiments of the inventive methods, RNA is converted to cDNA using reverse transcriptase following extraction of RNA from a bodily fluid and prior to amplification.
The methods of the invention are advantageously used for providing a diagnosis of, or as a predictive indicator for determining risk for a human of developing a proliferative, premalignant, neoplastic, or malignant disease comprising or characterized by cells expressing EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, hnRNP A2/B1 RNA or any combination thereof.
The methods of the invention are particularly useful for providing a diagnosis of or for identifying in animals, particularly humans, who are at risk for developing or who have developed malignancy or premalignancy of cells comprising epithelial tissues. Most preferably, malignant or premalignant diseases, conditions, or disorders advantageously detected or diagnosed using the methods of the invention are diseases or disorders of breast, ovaries, lung, cervix, colorectal, stomach, pancreas, bladder, endometrium, kidney, head and neck, and esophageal cancers, and premalignancies and carcinoma in-situ such as bronchial dysplasia, atypical hyperplasia of the breast, ductal carcinoma in-situ, colorectal adenoma, atypical endometrial hyperplasia, and Barrett""s esophagus.
In certain preferred embodiments of the methods of the invention, EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, hnRNP A2/B1 RNA or combinations thereof, or cDNA derived therefrom is amplified in a quantitative manner, thereby enabling quantitative comparison of said RNA present in a bodily fluid such as blood plasma or serum from a human. In these embodiments, the amount of extracellular EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, or hnRNP A2/B1 RNA or combinations thereof are detected in an individual and compared with a range of amounts of said extracellular RNA detected in the bodily fluid in a plurality of humans known to have a premalignant or malignant disease, or known to be free from a premalignant or malignant disease.
The invention further provides methods for identifying individuals having an EGF-, EGFr-, her-2/neu-, c-myc-, or hnRNP A2/B1-expressing malignancy or premalignancy, or a malignancy expressing any combination of said RNAs, thereby permitting rational, informed treatment options to be used for making therapeutic decisions. In particular, the methods of the invention are useful in identifying individuals having a premalignancy or malignancy that would benefit from a therapy directed at cells and tissues that express EGF, EGFr, her-2/neu-, c-myc-, or hnRNP A2/B1, such as monoclonal antibody therapy, anti-sense therapy, and vaccine therapy.
Another advantage of the use of the methods of the invention is that the methods can produce markers for assessing the adequacy of anticancer therapies such as surgical intervention, chemotherapy, biotherapy such as monoclonal antibody therapy or vaccines, anti-angiogenic therapy, and radiation therapy, and is also useful for determining whether additional or more advanced therapy is required. The invention therefore provides methods for developing a prognosis in such patients.
The methods of the invention also permit identification or analysis of EGF RNA, EGFr RNA, her-2/neu RNA, c-myc RNA, hnRNP A2/B1 RNA or any combination thereof, either qualitatively or quantitatively, in the blood or other bodily fluid of an animal, most preferably a human that has completed therapy, as an early indicator of relapsed cancer, impending relapse, or treatment failure.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.